B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates from a somatic DNA rearrangement process, called V(D)J recombination, that assembles the antigen receptor genes during lymphocyte development. This process proceeds in two phases: a cleavage phase during which the RAG1 and RAG2 proteins bind and cleave a pair of receptor gene coding segments at specific recombination signal sequences, and a joining phase during which the four DNA ends resulting from RAG-mediated cleavage are reorganized, processed and rejoined via the non-homologous end- joining (NHEJ) repair pathway. Emerging evidence suggests that the introduction, sensing, and repair of DNA breaks is actively coordinated through RAG protein interactions with other factors, but the mechanisms involved in these processes are not fully understood. We have identified two NHEJ repair factors, Ku70 and Ku80, and components of an E3 ubiquitin ligase complex as interaction partners with full-length RAG1. These findings lead us to speculate that the cleavage and joining phases of V(D)J recombination are connected and regulated by a ubiquitin modification pathway. In support of this possibility, we show that one of the E3 ubiquitin ligase components, called VprBP, is required for B cell development past the pro-B cell stage in mice. This developmental block is partially rescued by enforced Bcl2 expression, but under these conditions most emerging mature B cells express the Ig light chain. These data lead us to the specific hypothesis of this proposal: that VprBP is necessary for efficient and high-fidelity rearrangement of the large Igh and Igk loci, but not the more compact Igl locus. To test this hypothesis, we will establish the frequency and fidelity V(D)J recombination intermediates and products at the Igh, Igk, and Igl loci, as well as the accessibility of these loci, in VprBP-deficient B cells in the absence or presence of enforced Bcl2 expression. In addition, we will determine which step(s) of V(D)J recombination are specifically disrupted by loss of VprBP function using viral Abelson-transformed pre-B cell lines. Greater knowledge of how factors interacting with the RAG proteins, such as VprBP, guide the introduction, sensing, and repair of DNA breaks will improve our understanding of the mechanisms contributing to impaired and aberrant V(D)J recombination that underlie certain forms of immunodeficiency and lymphoid malignancy, respectively. PUBLIC HEALTH RELEVANCE: V(D)J recombination is a DNA rearrangement process that is responsible for generating a diverse population of B and T cells that together provide highly specific immunity to pathogenic microorganisms. V(D)J recombination is both essential and potentially dangerous to the host: defects in V(D)J recombination account for about 30% of human cases of severe combined immunodeficiency, yet certain forms of leukemia and lymphoma frequently bear molecular signatures implicating an aberrant V(D)J recombination event in their genesis. The research proposed here will investigate how VprBP, a component of an ubiquitin ligase complex that we have found associates with V(D)J recombinase, regulates V(D)J recombination during B cell development, which will improve our understanding of how this process contributes to B cell immune diversification and disease.